Mechanisms of Gonococcal Pilin Antigenic and Phase Variation

描述(申请人提供)：致病性奈瑟氏菌是人类特有的病原体，依赖于几种抗原变异系统在人类种群中持续定居并导致疾病。这一建议将进一步推动我们对淋球菌菌毛蛋白抗原变异的分子机制的研究。Pilin氨基酸序列的高频变化是由18个沉默的Pilin拷贝之一与单个表达的Pilin基因之间的基因转换反应所介导的。在过去的资助期间，我们已经鉴定了参与这一过程的大多数蛋白质，并证明了致病奈瑟菌携带二倍体染色体，这可能有助于基因转换。此外，我们还表明，Pilin抗原变异需要形成一种称为鸟嘌呤四元组(G4)的替代DNA结构，以及G4中一个小RNA的转录。在下一个资助阶段，我们将确定G4转录在Pilin抗原变异中的作用。结合G4结构的蛋白质将被鉴定出来，我们将测试这些蛋白质是否刺激G4结构的形成或溶解，和/或菌毛蛋白抗原变异的过程。我们还将确定G4结构是否通过阻止DNA复制来促进重组，以及特定的解旋酶是否阻止复制停滞。我们将探索细菌染色体中G4结构的形成，并确定G4结构是否与其他DNA序列相关联。最后，将确定各种部分功能丧失突变对菌毛蛋白抗原变异的影响。这些创新性研究的结果将对NeisSeries致病机制、抗原变异机制、DNA重组和复制以及多种细胞中不同DNA结构在分子过程中的作用等方面的研究产生重大影响。

DESCRIPTION (provided by applicant): The pathogenic Neisseria are obligate human pathogens that rely on several antigenic variation systems to continually colonize and cause disease in the human population. This proposal will further our studies into the molecular mechanisms used to allow pilin antigenic variation in Neisseria gonorrhoeae. High frequency changes in the pilin amino acid sequence are mediated by gene conversion reactions between one of 18 silent pilin copies and the single expressed pilin gene. In the past funding period, we have identified most of the proteins involved in this process and have demonstrated that the pathogenic Neisseria carry diploid chromosomes that may facilitate gene conversion. We have additionally shown that both the formation of an alternative DNA structure called a guanine quartet (G4), and the transcription of a small RNA within the G4 are required for pilin antigenic variation. In the next funding period we will determine the role of G4 transcription during pilin antigenic variation. Proteins that bind the G4 structure will be identified and we will test whethe these proteins stimulate G4 structure formation or dissolution, and/or the process of pilin antigenic variation. We will also determine whether the G4 structure acts to promote recombination by blocking DNA replication and whether specific helicases prevent a replication stall. We will probe for formation of the G4 structure within the bacterial chromosome and determine whether the G4 structure associates with other DNA sequences. Finally, the effect of various partial loss-of-function mutations on pilin antigenic variation will be determined. The results of these innovative studies will have great impact on the study of Neisserial pathogenesis, mechanisms of antigenic variation, DNA recombination and replication, and the role of alternative DNA structures on molecular processes in many cell types.